| Summary: | 碩士 === 國立交通大學 === 應用化學系碩博士班 === 105 === We have so far demonstrated the control of nucleation and crystal growth of hen egg-white lysozyme (HEWL) by laser trapping at a glass/solution interfacial layer in the supersaturated D2O solution. One of important findings in those studies is that firstly laser trapping forms a large-sized highly-concentrated domain of HEWL liquid-like clusters and then crystal nucleation and crystal growth take place in the dense cluster domain. Namely, it is important and indispensable to understand the formation dynamics and microscopic structure of the dense domain for developing the laser trapping-induced nucleation and crystal growth. In this research, we studied the formation dynamics of the cluster domain by measuring fluorescence of a probe dye molecule added in the HEWL D2O solution under laser trapping conditions.
RhB was used as a probe molecule and mixed with a HEWL D2O buffer solution. A continuous-wave near-infrared (NIR) laser beam was focused at a position 3 μm above a glass/solution interface of the solution. A 400-nm femtosecond laser was also introduced to the microscope as a fluorescence excitation light source and focused at a point 10 or 20 μm laterally away from the focal spot of the NIR laser. Fluorescence was detected with a single photon counting avalanche photodiode which was connected to a time-correlated single photon counting system. We measured the photon count and the fluorescence decay under laser trapping conditions.
The fluorescence intensity measured at points 10 and 20 μm away from the focus of the NIR laser was gradually increased during the laser trapping. After the trapping laser was switched off, the fluorescence intensity was decreased. We considered that the florescence intensity increase and decrease are ascribed to the formation and dissolution of a large-sized highly-concentrated cluster domain. The fluorescence lifetime showed tendency to become shorter during laser trapping, whereas it was gradually increased after turning off the trapping laser. The former was explained from the viewpoint of temperature distribution generated from the focal spot of the trapping laser. The latter was considered be due to association structure change of the dense cluster domain, which results in the spatial confinement of the crystallization in a millimeter-scale around the focal spot. Furthermore, we discussed the microscopic structure of the liquid-like clusters constituting the domain based on the fluorescence lifetime measurement. We believe that our study will be an important milestone for the microscopic study of laser trapping-induced crystallization and large dense domain formation.
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